The latent HIV-1 reservoir has become a major obstacle to virus eradication. The goal of eliminating this reservoir certainly requires the development of new therapeutic approaches other than the current antiretroviral therapy (ART). To develop one, a clear understanding of how latent HIV-1 is formed in vivo is necessary. Several primary cell models have been developed to study the mechanisms of viral reactivation from latency. However, it is evident that none of the current cell models closely recapitulates the mechanisms governing proviral latency in vivo. Therefore, developing a new in-vitro model which mimics natural proviral latency would be important. The objective of this R03 application is to develop a cell model of HIV-1 latency with advanced features that are missing in the current models, like the addition of cytotoxic T cell (CTL) pressure, the use of primary HIV strain, etc. The underlying hypothesis is that the inclusion of these features into a cell model wil closely mimic proviral latency that occurs in vivo. We will use CD8 T cells gene-modified with CD4-CD3q chimeric antigen receptor (namely, CD4CAR) as HIV-specific CTLs and the molecular clone-derived primary wild-type HIV strain HIVJRCSF to infect target cells in the model. In Specific Aim 1, we will develop a primary cell model to generate latently infected cells (LICs) by infecting activated CD4 T cells with HIVJRCSF and coculturing them with CD4CAR+CD8 T cells. Then we will characterize these LICs to verify how closely they represent their in-vivo counterparts. Once this model is developed, we will pursue our long-term goal to find unique cell surface markers expressed on LICs in subsequent studies. This will be followed by designing new approaches to target LICs through these markers for elimination in vivo. In Specific Aim 2, we will determine if noninduced proviruses are also generated in this model. If so, we will characterize these viruses to elucidate the mechanisms of their formation in vivo. It is our expectation that during this project period, we will develop the intended cell mode of proviral latency as an important tool for generating critical new information in future studies.